Compositions and methods for osteogenic gene therapy

Abstract

The present disclosure provides compositions and methods for increasing bone growth and/or enhancing wound healing, for example, fracture repair. The disclosure provides recombinant nucleic acids useful for promoting bone growth. For example, the disclosure provides recombinant nucleic acids that encode a fibroblast growth factor-2 (FGF-2) analog. The disclosure also provides vectors and cells incorporating these nucleic acids, as well as FGF-2 analogs encode by them. The disclosure also provides a mouse system of bone marrow transplantation and methods for producing as well as methods for using the system. Methods for inducing division and/or inducing differentiation of a hematopoietic stem cell are also provided, as are methods for enhancing bone growth and/or wound repair (for example, fracture repair).

We claim: 
     
       1. A method of identifying an agent that modulates bone growth, maintenance and repair, the method comprising:
 harvesting at least one hematopoietic stem cell; 
 transducing and expressing in the at least one mouse hematopoietic stem cell at least one heterologous nucleic acid comprising a polynucleotide sequence that encodes an osteogenic growth factor comprising a secretable analog of human fibroblast growth factor-2 (FGF-2) encoded by a nucleic acid sequence comprising in a 5′ to 3′ direction: a polynucleotide sequence that encodes a bone morphogenetic protein 2/4 (BMP2/4) hybrid secretion signal peptide sequence and a polynucleotide sequence that encodes a mature human FGF-2 polypeptide comprising a mutation that results in the substitution of an alanine for a cysteine in amino acid position 70 of FGF-2 and a mutation that results in the substitution of an asparagine for a cysteine in amino acid position 88 of FGF-2; 
 introducing the at least one hematopoietic stem cell or a progeny thereof into an immunologically compatible mouse, wherein expression of the secretable analog of human FGF-2 produces an increase in serum level of FGF-2 as compared to immunologically compatible mice engrafted with hematopoietic stem cells transduced with the BMP2/4 hybrid secretion signal peptide sequence and a wild type FGF-2-control; 
 administering an agent to the mouse; and 
 screening for modulation of FGF-2 secretion, wherein an increase in FGF-2 secretion identifies an agent that increases bone growth. 
 
     
     
       2. The method of  claim 1 , wherein the hematopoietic stem cell is a pluripotent hematopoietic stem cell. 
     
     
       3. The method of  claim 2 , wherein the hematopoietic stem cell expresses CXCR4. 
     
     
       4. The method of  claim 2 , wherein the hematopoietic stem cell is a Sca-1 +  cell. 
     
     
       5. The method of  claim 1 , wherein the at least one heterologous nucleic acid comprising the polynucleotide sequence that encodes an osteogenic growth factor is introduced into the hematopoietic stem cell by electroporation, biolistic transformation, lipid mediated transformation, or transfection of the cell with a viral vector comprising the heterologous nucleic acid. 
     
     
       6. The method of  claim 5 , wherein the viral vector is an adenovirus vector, an adeno-associated virus vector, a murine leukemia virus vector or a lentivirus vector. 
     
     
       7. The method of  claim 6 , wherein the viral vector is a replication defective viral vector. 
     
     
       8. The method of  claim 1 , wherein the polynucleotide sequence that encodes an osteogenic growth factor is operably linked to a transcription control sequence comprising a regulatable promoter. 
     
     
       9. The method of  claim 8 , wherein the transcription control sequence comprises a tetracycline regulatable promoter.